The present invention relates to the field of polymer synthesis and the use of polymer libraries for biological screening. More specifically, in one embodiment the invention provides arrays of diverse double-stranded oligonucleotide sequences. In another embodiment, the invention provides arrays of conformationally restricted probes, wherein the probes are held in position using double-stranded DNA sequences as scaffolding. Libraries of diverse unimolecular double-stranded nucleic acid sequences and probes may be used, for example, in screening studies for determination of binding affinity exhibited by binding proteins, drugs, or RNA.
Methods of synthesizing desired single stranded DNA sequences are well known to those of skill in the art. In particular, methods of synthesizing oligonucleotides are found in, for example, Oligonucleotide Synthesis: A Practical Approach, Gait, ed., IRL Press, Oxford (1984), incorporated herein by reference in its entirety for all purposes. Synthesizing unimolecular double-stranded DNA in solution has also been described. See, Durand, et al. Nucleic Acids Res. 18:6353-6359 (1990) and Thomson, et al. Nucleic Acids Res. 21:5600-5603 (1993), the disclosures of both being incorporated herein by reference.
Solid phase synthesis of biological polymers has been evolving since the early "Merrifield" solid phase peptide synthesis, described in Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963), incorporated herein by reference for all purposes. Solid-phase synthesis techniques have been provided for the synthesis of several peptide sequences on, for example, a number of "pins." See e.g., Geysen et al., J. Immun. Meth. 102:259-274 (1987), incorporated herein by reference for all purposes. Other solid-phase techniques involve, for example, synthesis of various peptide sequences on different cellulose disks supported in a column. See Frank and Doring, Tetrahedron 44:6031-6040 (1988), incorporated herein by reference for all purposes. Still other solid-phase techniques are described in U.S. Pat. No. 4,728,502 issued to Hamill and WO 90/00626 (Beattie, inventor).
Each of the above techniques produces only a relatively low density array of polymers. For example, the technique described in Geysen et al. is limited to producing 96 different polymers on pins spaced in the dimensions of a standard microtiter plate.
Improved methods of forming large arrays of oligonucleotides, peptides and other polymer sequences in a short period of time have been devised. Of particular note, Pirrung et al., U.S. Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070) and Fodor et al., PCT Publication No. WO 92/10092, all incorporated herein by reference, disclose methods of forming vast arrays of peptides, oligonucleotides and other polymer sequences using, for example, light-directed synthesis techniques. See also, Fodor et al., Science, 251:767-777 (1991), also incorporated herein by reference for all purposes. These procedures are now referred to as VLSIPS.TM. procedures.
In the above-referenced Fodor et al., PCT application, an elegant method is described for using a computer-controlled system to direct a VLSIPS.TM. procedure. Using this approach, one heterogenous array of polymers is converted, through simultaneous coupling at a number of reaction sites, into a different heterogenous array. See, U.S. Pat. No. 5,384,261 and U.S. application Ser. No. 07/980,523, the disclosures of which are incorporated herein for all purposes.
The development of VLSIPS.TM. technology as described in the above-noted U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092, is considered pioneering technology in the fields of combinatorial synthesis and screening of combinatorial libraries. More recently, patent application Ser. No. 08/082,937, filed Jun. 25, 1993 now abandoned, describes methods for making arrays of oligonucleotide probes that can be used to check or determine a partial or complete sequence of a target nucleic acid and to detect the presence of a nucleic acid containing a specific oligonucleotide sequence.
A number of biochemical processes of pharmaceutical interest involve the interaction of some species, e.g., a drug, a peptide or protein, or RNA, with double-stranded DNA. For example, protein/DNA binding interactions are involved with a number of transcription factors as well as tumor suppression associated with the p53 protein and the genes contributing to a number of cancer conditions.